This application addresses broad Challenge Area (06) Enabling Technologies, 06-NS-106: Validating new methods to study brain connectivity. We propose to test a new method that provides substantial improvement over previous Cre-conditional viral tracers. The technology combines the Brainbow multicolor cell marking technology with the retrograde, circuit tracing properties of pseudorabies virus (PRV), a neuroinvasive alpha herpesvirus. We have constructed a prototype PRV Brainbow virus called PRV263 that we propose will enable simultaneous identification of distinct chains of neurons projecting to a phenotypically defined population of neurons, and promises to provide predictive data on the strength of different connections among those neurons. Importantly, these PRV Brainbow tracers will have distinct advantages over present tracers. Our concept takes advantage of conditional, site-specific recombination of the genome of a DNA virus to produce multiple reporters so that neurons upstream (presynaptic) of a Cre recombinase (Cre) expressing neuron will be a different color from the Cre-expressing neuron. This novel concept will be expanded to produce second generation prototypes of PRV Brainbow tracers that do not rely on Cre-transgenic mice and can be used in the many mammalian species susceptible to PRV infection. A third generation prototype will be constructed that not only marks circuits, but also reports on neuronal activity. In this latter concept, the PRV Brainbow virus also will include a genetically encoded calcium indicator that fluoresces when calcium is bound. As viral tracing of neural circuitry has become an essential tool in the neuroscience community, our new tracers will be immediately applicable for many ongoing fundamental research projects in neuroscience in a variety of animals. These new tools have promise to reveal detailed functional insights into neural circuit organization that have not been possible to achieve in the past. Viral tracing of neural circuitry has become an essential tool in the neuroscience community. The new, robust viral tracers that will result from our work have promise to reveal detailed functional insights into trans-neuronal spread of herpesviruses, as well as neural circuit organization that have not been possible to achieve in the past. These neural tracers would be powerful tools to elucidate brain micro-circuitry, providing a better understanding of nervous system functions.